TY - JOUR A1 - Deutschmann, Claudia A1 - Roggenbuck, Dirk A1 - Schierack, Peter A1 - Rödiger, Stefan T1 - Autoantibody testing by enzyme-linked immunosorbent assay-a case in which the solid phase decides on success and failure JF - Heliyon N2 - Background: The enzyme-linked immunosorbent assay (ELISA) is an indispensable tool for clinical diagnostics to identify or differentiate diseases such as autoimmune illnesses, but also to monitor their progression or control the efficacy of drugs. One use case of ELISA is to differentiate between different states (e.g. healthy vs. diseased). Another goal is to quantitatively assess the biomarker in question, like autoantibodies. Thus, the ELISA technology is used for the discovery and verification of new autoantibodies, too. Of key interest, however, is the development of immunoassays for the sensitive and specific detection of such biomarkers at early disease stages. Therefore, users have to deal with many parameters, such as buffer systems or antigen-autoantibody interactions, to successfully establish an ELISA. Often, fine-tuning like testing of several blocking substances is performed to yield high signal-to-noise ratios.
Methods: We developed an ELISA to detect IgA and IgG autoantibodies against chitinase-3-like protein 1 (CHI3L1), a newly identified autoantigen in inflammatory bowel disease (IBD), in the serum of control and disease groups (n = 23, respectively). Microwell plates with different surface modifications (PolySorp and MaxiSorp coating) were tested to detect reproducibility problems.
Results: We found a significant impact of the surface properties of the microwell plates. IgA antibody reactivity was significantly lower, since it was in the range of background noise, when measured on MaxiSorp coated plates (p < 0.0001). The IgG antibody reactivity did not differ on the diverse plates, but the plate surface had a significant influence on the test result (p = 0.0005).
Conclusion: With this report, we want to draw readers' attention to the properties of solid phases and their effects on the detection of autoantibodies by ELISA. We want to sensitize the reader to the fact that the choice of the wrong plate can lead to a false negative test result, which in turn has serious consequences for the discovery of autoantibodies. KW - biochemistry KW - coatings KW - surface chemistry KW - immunology KW - proteins KW - laboratory medicine KW - clinical research KW - enzyme-linked immunosorbent KW - assay KW - biomarker discovery KW - reproducibility KW - solid-phase KW - autoantibody Y1 - 2020 U6 - https://doi.org/10.1016/j.heliyon.2020.e03270 SN - 2405-8440 VL - 6 IS - 1 PB - Elsevier CY - London [u.a.] ER - TY - GEN A1 - Choi, Youngeun A1 - Schmidt, Carsten A1 - Tinnefeld, Philip A1 - Bald, Ilko A1 - Rödiger, Stefan T1 - A new reporter design based on DNA origami nanostructures for quantification of short oligonucleotides using microbeads T2 - Postprints der Universität Potsdam : Mathematisch-naturwissenschaftliche Reihe N2 - The DNA origami technique has great potential for the development of brighter and more sensitive reporters for fluorescence based detection schemes such as a microbead-based assay in diagnostic applications. The nanostructures can be programmed to include multiple dye molecules to enhance the measured signal as well as multiple probe strands to increase the binding strength of the target oligonucleotide to these nanostructures. Here we present a proof-of-concept study to quantify short oligonucleotides by developing a novel DNA origami based reporter system, combined with planar microbead assays. Analysis of the assays using the VideoScan digital imaging platform showed DNA origami to be a more suitable reporter candidate for quantification of the target oligonucleotides at lower concentrations than a conventional reporter that consists of one dye molecule attached to a single stranded DNA. Efforts have been made to conduct multiplexed analysis of different targets as well as to enhance fluorescence signals obtained from the reporters. We therefore believe that the quantification of short oligonucleotides that exist in low copy numbers is achieved in a better way with the DNA origami nanostructures as reporters. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 705 KW - nucleic-acids KW - hybridization KW - microrna KW - flourescence KW - biomarkers KW - platform KW - particle KW - binding KW - array KW - gene Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-428271 SN - 1866-8372 IS - 705 ER - TY - JOUR A1 - Choi, Youngeun A1 - Schmidt, Carsten A1 - Tinnefeld, Philip A1 - Bald, Ilko A1 - Rödiger, Stefan T1 - A new reporter design based on DNA origami nanostructures for quantification of short oligonucleotides using microbeads JF - Scientific Reports N2 - The DNA origami technique has great potential for the development of brighter and more sensitive reporters for fluorescence based detection schemes such as a microbead-based assay in diagnostic applications. The nanostructures can be programmed to include multiple dye molecules to enhance the measured signal as well as multiple probe strands to increase the binding strength of the target oligonucleotide to these nanostructures. Here we present a proof-of-concept study to quantify short oligonucleotides by developing a novel DNA origami based reporter system, combined with planar microbead assays. Analysis of the assays using the VideoScan digital imaging platform showed DNA origami to be a more suitable reporter candidate for quantification of the target oligonucleotides at lower concentrations than a conventional reporter that consists of one dye molecule attached to a single stranded DNA. Efforts have been made to conduct multiplexed analysis of different targets as well as to enhance fluorescence signals obtained from the reporters. We therefore believe that the quantification of short oligonucleotides that exist in low copy numbers is achieved in a better way with the DNA origami nanostructures as reporters. KW - nucleic-acids KW - hybridization KW - microrna KW - flourescence KW - biomarkers KW - platform KW - particle KW - binding KW - array KW - gene Y1 - 2019 U6 - https://doi.org/10.1038/s41598-019-41136-x SN - 2045-2322 IS - 9 PB - Macmillan Publishers Limited CY - London ER - TY - JOUR A1 - Schmidt, Carsten A1 - Schierack, Peter A1 - Gerber, Ulrike A1 - Schroeder, Christian A1 - Choi, Youngeun A1 - Bald, Ilko A1 - Lehmann, Werner A1 - Rödiger, Stefan T1 - Streptavidin homologues for applications on solid surfaces at high temperatures JF - Langmuir N2 - One of the most commonly used bonds between two biomolecules is the bond between biotin and streptavidin (SA) or streptavidin homologues (SAHs). A high dissociation constant and the consequent high-temperature stability even allows for its use in nucleic acid detection under polymerase chain reaction (PCR) conditions. There are a number of SAHs available, and for assay design, it is of great interest to determine as to which SAH will perform the best under assay conditions. Although there are numerous single studies on the characterization of SAHs in solution or selected solid phases, there is no systematic study comparing different SAHs for biomolecule-binding, hybridization, and PCR assays on solid phases. We compared streptavidin, core streptavidin, traptavidin, core traptavidin, neutravidin, and monomeric streptavidin on the surface of microbeads (10-15 mu m in diameter) and designed multiplex microbead-based experiments and analyzed simultaneously the binding of biotinylated oligonucleotides and the hybridization of oligonucleotides to complementary capture probes. We also bound comparably large DNA origamis to capture probes on the microbead surface. We used a real-time fluorescence microscopy imaging platform, with which it is possible to subject samples to a programmable time and temperature profile and to record binding processes on the microbead surface depending on the time and temperature. With the exception of core traptavidin and monomeric streptavidin, all other SA/SAHs were suitable for our investigations. We found hybridization efficiencies close to 100% for streptavidin, core streptavidin, traptavidin, and neutravidin. These could all be considered equally suitable for hybridization, PCR applications, and melting point analysis. The SA/SAH-biotin bond was temperature sensitive when the oligonucleotide was mono-biotinylated, with traptavidin being the most stable followed by streptavidin and neutravidin. Mono-biotinylated oligonucleotides can be used in experiments with temperatures up to 70 degrees C. When oligonucleotides were bis-biotinylated, all SA/SAH-biotin bonds had similar temperature stability under PCR conditions, even if they comprised a streptavidin variant with slower biotin dissociation and increased mechanostability. Y1 - 2020 U6 - https://doi.org/10.1021/acs.langmuir.9b02339 SN - 0743-7463 VL - 36 IS - 2 SP - 628 EP - 636 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Czarnecka, Malgorzata A1 - Weichelt, Ulrike A1 - Rödiger, Stefan A1 - Hanack, Katja T1 - Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA JF - International Journal of Molecular Sciences N2 - The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures. KW - antibody KW - camelid antibody KW - heavy-chain-only antibody KW - miRNA KW - nucleic acids KW - novel biomarkers Y1 - 2022 U6 - https://doi.org/10.3390/ijms23116275 SN - 1422-0067 VL - 23 SP - 1 EP - 18 PB - MDPI CY - Basel, Schweiz ET - 11 ER - TY - GEN A1 - Czarnecka, Malgorzata A1 - Weichelt, Ulrike A1 - Rödiger, Stefan A1 - Hanack, Katja T1 - Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1274 KW - antibody KW - camelid antibody KW - heavy-chain-only antibody KW - miRNA KW - nucleic acids KW - novel biomarkers Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-569142 SN - 1866-8372 SP - 1 EP - 18 ER -